Mutagenic treatments

N-nitrosomethylurea (NMU). Cells were treated with 0.1 or 0.5% solutions of NMU in phosphate-citrate buffer for 30 min, 2, 3,4 and 5 h. 

Dimethylsulfate. We used a 1 6000 dilution (0.017% solution). Treatment time was 12 to 16 h. Alternatively, cells were treated with the mutagen vapor according to the method of Kupenov (1974). In this case, a small vessel containing the mutagen liquid was placed on the bottom of a tube that contained a thin layer of cells deposited on the walls by vacuum drying. The action of the DMS vapor was stopped by removing the vessel from the tube. 

N-methyl-N -nitro-N -nitrosoguanidine (NTG). NTG is unstable in solution. At pH 7.0 nitrosoguanidine is converted to diazomethane, at pH 5.0 to nitric acid. Diazomethane and nitric acid also are mutagenic but less effective than NTG. NTG is relatively stable at pH 5.5-6.5. Therefore, cells were treated with 0.05% solution of NTG in phosphate buffer at pH 6.0. Treatment time was 20, 40 and 60 min. 

Cell survival, presence of morphological mutants and variability of Co incorporation were determined. For each variant the results were statistically analyzed by calculating the mean value (x), standard deviation (cr) and covariation (CV) (Vorobjeva et al., 1973). 

The yield of positive mutants (activity x + 3cr) was 1.2%. As a result of the DMS treatment, variants 2 and 1 were isolated, superior to the parental strain in vitamin B12 production by 30% (Table 2.5). 

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From this one ancestral fungus each penicillin manufacturer has evolved a particular production strain by a series of mutagenic treatments, each followed by the selection of improved variants. These selected variants have proved capable of producing amounts of penicillin far greater than those produced by the wild strain, especially when fermented on media under particular control conditions developed in parallel with the strains. These strain selection procedures have become a fundamental feature of industrial biotechnology. 

Because the occurrence of mutation is rare even after mutagen treatment, the specific locus test is the ultimate study of mutation, requiring many thousands of offspring to be scored, plus significant resources of time, space, and animal... 

A different STA-producing strain. Strep, longisporqflavus, was subjected to a mutagenic treatment by UV irradiation. This work led to the isolation of mutants M13 and M14, which accumulated STA aglycone (9, also known as K252c) and 3 -demethoxy-3 -hydroxy-STA (8), respectively [18,19]. The latter compound was converted into STA by cultures of the M13 mutant, and hence it was proposed as the last intermediate in STA biosynthesis [20]. 

If neoplastic plasma cells can sometimes stimulate osteogenesis, which they more usually inhibit, they might also stimulate erythroid cells, which again it seems they most commonly inhibit. This could be one view of paraproteins associated with polycythemia and even erythro-leukemia. Another view is that the general myeloproliferative state either per se or as a result of mutagenic treatment has evolved a mono-clone of plasma cells. A third view (see Section 7.6.4) can explain some erythroleukemia as misinterpretation of light microscopy. 

In phenylamide-resistant strains, phenylamides are not able to inhibit ribosomal RNA synthesis, as is indicated by the inability of metalaxyl to interfere with uridine incorporation in a phenylamide-resistant strain of P. megasperma f. sp. megasperma obtained in the laboratory after mutagenic treatment (14) and a phenylamide-resistant strain of Phytophthora infestans (Table III) originating from a potato field (15). 

The development of conjugation systems for gene transfer in the fastgrowing strains of Rhizobium has provided a method for studying genetic lesions in symbiotically defective mutants isolated directly from nodules or produced by mutagenic treatments (Maier and Brill, 1978 Beringer et al.. 

A. Use of a Two-Component Heterokaryon to Measure the Genetic Effects of Mutagenic Treatment... 

Atwood and Mukai (1954) have also shown that a comparison of the inactivation kinetics of the heterokaryotic fraction of conidia with the two homokaryotic fractions provides information on the mechanism of inactivation, whether nuclear or cytoplasmic. If the mutagenic treatment kills the cells by inactivation of nuclei, the proportion of heterokaryotic survivors among the total survivors will decrease very rapidly as the survival fraction of cells decreases. However, if the mutagenic treatment kills the cells by inactivation of the c) oplasm, the fraction of heterokaryotic survivors among the total survivors will remain constant. 

One of the anticipated applications of the cultured cell systems was for experimental estimation of mammalian and human mutation rates. Cell cultures have been used extensively to study chromosomal damage resulting from mutagenic treatments (this aspect is treated in a separate chapter). On the other hand, the spontaneous mutation rates of several phenotypes have been estimated in a few established lines of mammalian cells (Lieber-man and Ove, 1959 Szybalski, 1964 Chu et al., 19696). Early attempts to... 

FIGURE 2. Experimental schemes for induction of mutations in mammalian cell cultures. A. Cells are inoculated and allowed to attach to the plates before mutagen treatment. B. Cells are treated with mutagen before inoculation. The schemes are applicable to either chemical or physical mutagenesis. 

Expression time (hr) Mutagen treatment Plating efficiency Percent survival Mutation frequency per 10 survivors... 

Mutagen treatment 8-azaguanine C g/ml) Definitive colonies All colonies... 

When the males are treated, the females are transferred twice daily. The eggs are counted hatchability and stages of embryonic death are recorded 30-40 hr later. After 7 more days, female offspring are collected as virgins for F testing. Adult survival and stages of larval and pupal death are recorded. These data are used to determine the frequencies of dominant lethality induced hy the mutagenic treatment. 

Any female found to be heterozygous for a genetic alteration is out-crossed to a male with a sex allele that differs from either of hers this cross prevents production of diploid male progeny (Fig. 2, part 7). Egg counts, hatchability, and adult survival are collected. Analysis of these data yields the separate frequencies of recessive lethality and inherited partical sterility induced hy the mutagenic treatment cf. Whiting et aL, 1968). 

The incidence of late deaths is the same in the controls and after mutagenic treatment (Russell et aLy 1954 Bateman, 1958a Lyon et aLy 1964). It should therefore be possible to obtain better discrimination by separate recording of early and late deaths. A further refinement would be to make... 

Except for bands in the histone region, the most conspicuous acceptor of label for both untreated and treated samples was a species of 116,000 daltons. This protein is probably poly(ADP-ribose) polymerase. The histone region, which also shows a substantial increase in 32p-iabeling, is more complicated. Mutagen treatment resulted in the increased labeling of a series of equally spaced bands. The ladder of bands could represent modified histones with poly(ADP-ribose) chains of different lengths or free chains of poly(ADP-ribose). 

The alterations in DNA stmcture produced by mutagen treatment which... 

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